Compound preparation for resisting fish stress and application thereof

Abstract

The application relates to the technical field of anti-fish stress, in particular to an anti-fish stress compound preparation and application thereof. The compound preparation is prepared from 1-100 parts of angelica sinensis, 1-100 parts of rosemary and 1-100 parts of perilla leaf extract. The compound preparation can improve the anti-stress ability of the pelteobagrus fulvidraco under the condition of excessive feeding after temperature rapid change, relieve the body resistance decline of the pelteobagrus fulvidraco under the stress state caused by the excessive feeding, and improve the survival rate of breeding.

Description

Technical Field

[0001] This application relates to the field of anti-fish stress technology, specifically to a compound preparation for anti-fish stress and its application. Background Technology

[0002] Fish stress response refers to a non-specific physiological reaction in fish to abnormal stimuli (also known as stressors) from various environmental factors. Excessive or prolonged stress can harm the fish, leading to slow growth and development, decreased reproductive capacity, weakened immune function, and increased morbidity and mortality. Common stressors include: firstly, environmental factors such as water temperature, salinity, dissolved oxygen, ammonia, and pH; secondly, physical disturbances such as transportation, pond separation, and sex selection; and thirdly, biological factors such as overcrowding and pathogenic microbial invasion.

[0003] Common measures to reduce stress in fish include using aeration equipment, administering vitamins, adding zeolite, and using microbial preparations. However, during large-scale fish disease outbreaks, affected fish exhibit extremely high stress levels. For example, yellow catfish experience severe stress responses under the dual stress of rapid temperature changes and overfeeding, affecting their metabolic functions and further disrupting their internal homeostasis and reducing their disease resistance, making them more susceptible to bacterial and parasitic diseases. Conventional measures are often insufficient to combat these stress responses. Summary of the Invention

[0004] In view of this, the inventors of this application have creatively disclosed a compound preparation for combating fish stress, which can improve the stress resistance of yellow catfish under conditions of overfeeding following rapid temperature changes, alleviate the resulting decrease in the fish's resistance under stress, and thus improve the survival rate in aquaculture. Therefore, the embodiments of this application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose a compound preparation for resisting fish stress, which is prepared by comprising 1 to 100 parts by weight of Angelica sinensis, 1 to 100 parts by weight of rosemary and 1 to 100 parts by weight of perilla leaf extract.

[0006] In some embodiments, the raw materials of the compound preparation include 30 to 80 parts by weight of Angelica sinensis, 20 to 50 parts by weight of rosemary and 20 to 50 parts by weight of perilla leaf extract.

[0007] In some embodiments, the ingredients of the compound preparation include 40 parts by weight of Angelica sinensis, 30 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

[0008] In some embodiments, the ingredients of the compound preparation include 40 parts by weight of Angelica sinensis, 20 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

[0009] In some embodiments, the ingredients of the compound preparation include 40 parts by weight of Angelica sinensis, 30 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

[0010] In some embodiments, the ingredients of the compound preparation include 40 parts by weight of Angelica sinensis, 20 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

[0011] Secondly, the embodiments disclose a method for preparing a compound preparation for combating fish stress. The method includes adding the prescribed amounts of extracts of Angelica sinensis, rosemary, and perilla leaves to water and decocting at 80–100°C for 2–4 hours, filtering, concentrating and drying the filtrate, and obtaining the solid product as the compound preparation.

[0012] Thirdly, the embodiments disclose the application of the compound preparation described in the first aspect in the preparation of an anti-fish stress preparation.

[0013] In some embodiments, the application includes administering 0.3 to 1 ppm of the compound preparation to the water in which the fish grow.

[0014] Traditional Chinese medicine (TCM) has advantages such as wide availability, multiple targets, and no toxic side effects, and is increasingly being used in the prevention and treatment of aquatic diseases. To address this challenge, the inventors screened various TCM herbs and formulated compound prescriptions. They ultimately discovered that a compound of Angelica sinensis, rosemary, and Perilla frutescens leaf extracts in a 4:3:3 ratio could exert a good anti-stress effect and provide excellent protection for yellow catfish under the dual stress of rapid temperature changes and overfeeding in early spring.

[0015] Angelica sinensis mainly comprises volatile oils, flavonoids, amino acids, organic acids, and polysaccharides. Its properties are sweet, warm, and pungent, and it enters the heart, spleen, and liver meridians. It has the effects of nourishing blood and promoting blood circulation, anti-inflammation, anti-tumor, regulating menstruation and relieving pain, and enhancing immunity. Rosemary mainly comprises phenolic acids, flavonoids, terpenes, and essential oil compounds, possessing various pharmacological effects such as antioxidant, antibacterial, antitumor, anti-inflammatory, and antidepressant properties. Perilla leaves contain various bioactive substances, mainly categorized as volatile oils, flavonoids, phenolic acids, anthocyanins, polysaccharides, triterpenes, and steroidal compounds, exhibiting good therapeutic effects such as antitumor, antibacterial, anti-inflammatory, anti-influenza, anti-allergic, antioxidant, antidepressant, anticancer, detoxifying, sedative, and analgesic properties. Using Angelica sinensis as the main herb and rosemary and perilla leaf extracts as excipients, through optimized formulation, it exerts a good anti-stress effect. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0017] 1. Compound preparations

[0018] The prescribed amounts of Angelica sinensis, rosemary, and Perilla frutescens leaf extracts are added to water and decocted at 80–100°C for 2–4 hours. The mixture is then filtered, and the filtrate is concentrated and dried. The resulting solid is the compound preparation. The preparation of the Perilla frutescens leaf extract involves weighing 200g of dried Perilla frutescens leaves and refluxing them three times with 50% ethanol, each time for 3 hours. The residue is filtered, and the extracts are combined and concentrated under reduced pressure. The final product is then refrigerated for later use. The term "part" refers to any weight as a part, used only to define the weight ratio, not to specify a specific absolute weight.

[0019] Table 1 Raw materials for compound preparations

[0020] Serial Number Angelica sinensis (portion) Rosemary (servings) Perilla leaf extract (per serving) Formulation 1 40 20 10 Formulation 2 40 20 20 Formulation 3 40 20 30 Formulation 4 40 30 10 Formulation 5 40 30 20 Formulation 6 40 30 30

[0021] Test 1

[0022] 1. Test Materials and Methods

[0023] The yellow catfish, weighing approximately 10g, was purchased from a fish farmer in Jiangxia District, Wuhan City.

[0024] The experiment was conducted in a glass aquarium (57.8cm × 38.2cm × 22.7cm). The fish were randomly divided into eight groups: a control group (no temperature change, no medication), a negative control group (temperature change, no medication), and six test groups (temperature change, medication). Each group had three replicates, with ten fish per tank. During the initial holding period, the water temperature was 15℃. After the experiment began, each test group was given the corresponding compound preparation at 0.5 ppm in the water, and the water temperature was increased by 1℃ every 4 hours, reaching 25℃ within 48 hours. The water temperature in the control group remained unchanged, while the water temperature in the negative control group was increased to 25℃ within 48 hours. Samples were taken at 48 hours. Three fish were randomly selected from each tank, and blood was collected from the tail vein. After standing for 30 minutes, the blood was incubated overnight at 4℃, and the serum samples were separated by centrifugation at 3000 rpm for 10 minutes to determine the cortisol content in the serum.

[0025] 2. Test Results and Analysis

[0026] Table 2 shows that the cortisol content was as follows: negative control group > preparation 1 group > preparation 4 group > preparation 2 group > preparation 3 group > preparation 5 group > preparation 6 group > control group. Among these, there were no significant differences between preparation 1 and preparation 4 and the negative control group (P > 0.05), significant differences between preparation 2, preparation 3, preparation 5, and preparation 6 and the negative control group (P < 0.05), and no significant difference between preparation 6 and the control group (P > 0.05).

[0027] Table 2. Serum cortisol levels in yellow catfish from each group. Superscript labels indicate significant differences.

[0028] Serial Number Cortisol content (ng / mL) Formulation 1 <![CDATA[149.35±12.49 a ]]> Formulation 2 <![CDATA[127.36±14.27 b ]]> Formulation 3 <![CDATA[120.94±10.54 b ]]> Formulation 4 <![CDATA[136.31±9.93 a ]]> Formulation 5 <![CDATA[114.28±17.64 c ]]> Formulation 6 <![CDATA[106.53±13.58 d ]]> control group <![CDATA[98.35±6.27 d ]]> negative control group <![CDATA[156.42±10.54 a ]]>

[0029] From a physiological perspective, fish stress responses are generally classified into primary, secondary, and tertiary stress responses based on their occurrence process. The primary stress response mainly involves the recognition of the stressor and the activation of biological defenses. It is a response at the level of the nervous and endocrine systems, activating two stress response systems primarily the sympathetic-chromaffin system and the hypothalamic-pituitary-renal axis, leading to an increase in blood levels of stress hormones such as catecholamines and cortisol. Cortisol is widely recognized as a good indicator for evaluating fish stress responses due to its good stability and the positive correlation between the magnitude and duration of its increase and the intensity and duration of the stressor's effect. In this test, under the influence of a sharp increase in temperature, the serum cortisol content of yellow catfish in the negative control group significantly increased (P < 0.05). After medication, the serum cortisol content of yellow catfish in preparations 2, 3, 5, and 6 significantly decreased (P < 0.05), indicating that the preparations improved the stress response of yellow catfish. Among these, the serum cortisol content of yellow catfish in preparation 6 showed no significant difference from the control group (P > 0.05), exhibiting the best anti-stress effect. Therefore, the compound preparation provided in this application has a significant anti-stress effect.

[0030] Test 2

[0031] 1. Test Materials and Methods

[0032] Yellow catfish, weighing approximately 10g, were purchased from a fish farmer in Jiangxia District, Wuhan City. To simulate sudden temperature changes in spring, two rapid temperature change groups were designed: one group experienced a rapid temperature rise (from 15℃ to 25℃), and the other a rapid temperature drop (from 25℃ to 15℃). Both groups were further divided into normal feeding, overfeeding, and medication overfeeding groups (adding compound preparation 6 at 0.5 ppm to the water). Each group had three replicates, with 20 fish per replicate. Fish in the rapid temperature drop group were temporarily held at 25℃ for 7 days without feeding, and the water temperature was then lowered to 15℃ within 48 hours after the temporary holding period. Fish in the rapid temperature rise group were temporarily held at 15℃ for 7 days without feeding, and the water temperature was then raised to 25℃ within 48 hours. Normal feeding amount: 1.5% of the fish's body weight at low temperature (15℃) and 3% at suitable temperature (25℃); Overfeeding: 2.5% of the fish's body weight at low temperature and 4.5% at suitable temperature. Samples were taken at 0h, 24h, and 48h after the start of the experiment. Three experimental fish were randomly selected from each tank, and blood was collected from the tail vein. After standing for 30 minutes, the samples were centrifuged at 3000 rpm for 10 minutes to separate serum samples. Lysozyme activity, alkaline phosphatase activity, and cortisol concentration in the serum were detected using standard methods.

[0033] 2. Test Results and Analysis

[0034] As shown in Table 3, in the rapidly increasing temperature group, the cortisol content showed an increasing trend in all groups. The drug-treated group was slightly higher than the normal feeding group and lower than the overfeeding group. Lysozyme activity increased over time. The overfeeding group had higher activity than the normal feeding group, while the drug-treated group had slightly higher activity than the normal feeding group and lower activity than the overfeeding group. Alkaline phosphatase activity first increased and then decreased in the normal feeding group, showed a decreasing trend in the overfeeding group, and first increased and then decreased in the drug-treated group, but the differences in each time period were not significant (P>0.05).

[0035] In the rapid cooling group, cortisol levels increased in all groups, with the medication group slightly higher than the normal feeding group and lower than the overfeeding group; lysozyme activity first increased and then decreased over time, with the medication group higher than the normal feeding group and lower than the overfeeding group at 24h, and higher in the medication group than the normal feeding group at 48h; alkaline phosphatase activity first increased and then decreased in the normal feeding group, decreased in the overfeeding group, and first increased and then decreased in the medication group, but the differences at each time point were not significant (P>0.05).

[0036] Studies have shown that cortisol levels in fish increase under stress conditions, and the severity of stress can be indicated by the degree and duration of this increase. Therefore, cortisol levels can be an important physiological indicator of stress levels. In different temperature-changing groups, overfeeding led to an increase in cortisol levels. The cortisol levels in the medication group were lower than those in the overfeeding group, indicating that the compound preparation can alleviate the stress caused by overfeeding after a rapid temperature change.

[0037] Lysozyme is a non-toxic alkaline protein widely found in fish mucus, liver, digestive tract secretions, serum, and lysosomes, and is an important non-specific immune factor. Alkaline phosphatase catalyzes the hydrolysis of phosphate monoesters and the conversion of phosphate groups, playing a crucial role in the immune defense response of organisms. Therefore, lysozyme and alkaline phosphatase can be used to evaluate the disease resistance of an organism. The results show that overfeeding led to increased lysozyme activity in the warming group, while lysozyme activity in the cooling group initially increased and then decreased. Alkaline phosphatase activity decreased in both the warming and cooling groups, indicating that the stress response from overfeeding after rapid temperature changes reduces the resistance of yellow catfish. The lysozyme and alkaline phosphatase activities in the medication group were higher than those in the overfeeding group, indicating that the compound preparation can improve the stress resistance of yellow catfish and alleviate the decrease in resistance under the stress of overfeeding after rapid temperature changes.

[0038] Table 3. Effects of the compound preparation on improving serum biochemical parameters of yellow catfish under the dual stress of temperature and overfeeding.

[0039]

[0040] Therefore, the compound preparation provided in this application can improve the stress resistance of yellow catfish under conditions of excessive feeding after rapid temperature changes, and alleviate the decline in the body's resistance under stress caused by this.

[0041] Test 3

[0042] 1. Materials and Methods

[0043] The experiment was conducted at a yellow catfish farm in Jiangxia District, Wuhan City. During the spring temperature fluctuation period (April-May), the experimental ponds were treated with a 0.5 ppm formulation every other day for one month, while the control ponds were not treated. The survival rate of the yellow catfish was observed during the period. At the end of the experiment, 10 fish from each group were taken, and blood was collected from the tail vein. After standing for 30 minutes, the blood samples were separated by centrifugation at 3000 rpm for 10 minutes, and relevant indicators were measured.

[0044] 2. Test Results and Analysis

[0045] During the experiment, a total of 32 fish died in the test group and 211 fish died in the control group. The effectiveness rate of reducing spring mortality in farmed yellow catfish reached 84.8% ((211-32)÷211=84.8%).

[0046] Table 3. Changes in serum biochemical indicators

[0047] Cortisol (ng / mL) Lysozyme (U / mL) Alkaline phosphatase (U / L) control group 134.26±1.33 243.48±12.47 18.39±0.52 Test group 104.53±5.68* 324.72±16.2* 27.41±3.85*

[0048] Note: "*" indicates a significant difference, P < 0.05

[0049] Table 3 shows that the cortisol level in the test group was significantly lower than that in the control group (P < 0.05), while the activities of lysozyme and alkaline phosphatase in the test group were significantly higher than those in the control group (P < 0.05). When fish are under prolonged stress, changes such as decreased growth rate, reproductive capacity, and disease resistance occur at the individual or group level. Studies have shown that cortisol levels in fish increase under stress conditions, and the severity of stress can be expressed by the degree and duration of this increase. Therefore, cortisol level can be an important physiological indicator of stress level. The decrease in cortisol levels in the test group indicates that the medication relieved the stress.

[0050] Lysozyme is a non-toxic, alkaline protein widely found in fish mucus, liver, digestive tract secretions, serum, and lysosomes, and is an important non-specific immune factor. Alkaline phosphatase catalyzes the hydrolysis of phosphate monoesters and the conversion of phosphate groups, playing a crucial role in the immune defense response of organisms. Therefore, lysozyme and alkaline phosphatase can be used to evaluate an organism's disease resistance. The serum lysozyme and alkaline phosphatase activities in the test group of yellow catfish were both higher than those in the control group, indicating that the disease resistance of the treated yellow catfish was enhanced.

[0051] Therefore, the compound preparation provided in this application can improve the stress resistance of farmed yellow catfish and increase the survival rate of farmed fish.

[0052] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A compound preparation for combating fish stress, prepared from the following raw materials in parts by weight: 40 parts Angelica sinensis, 20-30 parts Rosemary and 20-30 parts Perilla frutescens leaf extract; in, The perilla leaf extract was obtained by reflux extraction of perilla leaves with 50% ethanol by volume.

2. The compound preparation according to claim 1, wherein the raw materials of the compound preparation are composed of 40 parts by weight of Angelica sinensis, 30 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

3. The compound preparation according to claim 1, wherein the raw materials of the compound preparation are composed of 40 parts by weight of Angelica sinensis, 20 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

4. The compound preparation according to claim 1, wherein the raw materials of the compound preparation are composed of 40 parts by weight of Angelica sinensis, 30 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

5. The compound preparation according to claim 1, wherein the raw materials of the compound preparation are composed of 40 parts by weight of Angelica sinensis, 20 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

6. A method for preparing a compound preparation for relieving fish stress, comprising mixing the raw materials of the compound preparation as described in any one of claims 1 to 5, adding water and decocting at 80 to 100°C for 2 to 4 hours, filtering, concentrating and drying the filtrate, and obtaining the solid as the compound preparation.

7. The use of the compound preparation according to any one of claims 1 to 5 in the preparation of an anti-fish stress formulation.

8. The application according to claim 7, comprising administering 0.3 to 1 ppm of the compound preparation to the water in which the fish grow.

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